Protein kinases are enzymes which catalyze protein phosphorylation, a key cellular regulatory mechanism which is frequently deregulated in human diseases. Consequently, protein kinases represent interesting targets for the pharmaceutical industry in its search for new therapeutic agents. 518 Human protein kinase genes have been identified in the human kinome (Manning G. et al., Science, 2002, 298, 1912-34). It is well known that protein kinases are key elements in intracellular signaling pathways that control many physiological processes. Most kinases act on both serine and threonine, others act on tyrosine, and a number (dual-specificity kinases) act on all three.
Dual-specificity tyrosine-regulated kinases (DYRKs) comprise a family of protein kinases within the CMGC group of the eukaryotic kinome (CMGC: cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs), glycogen synthase kinases (GSKs), and CDK-like kinases (CLKs). The DYRK family comprises five members in humans, DYRK1A, DYRK1B, DYRK2, DYRK3, and DYRK4 (Becker et al., J. Biol. Chem., 1998, 273(40), 25893-902).
DYRK1A may play a significant role in a signaling pathway regulating cell proliferation and may be involved in brain development.
Expression of DYRK1A is detected in several regions of the central nervous system, from development to adulthood, especially in the cortex, hippocampus and cerebellum. Dyrk1A knock-out mice are embryonic lethal and transgenic mice overexpressing Dyrk1A display learning and memory deficiencies. The human DYRK1A gene has been implicated in the pathogenesis of Down syndrome due to its location on the Down syndrome (DS) critical region of the human chromosome 21 and is present in three copies in DS patients. Trisomy-driven overexpression in DS patients has been demonstrated and DYRK1A overexpression in DS is also associated with early Alzheimer's disease (AD) phenotype observed in DS patients.
In DS, the pathogenetic role of enhanced activity of DYRK1A in both neurodevelopment and neurodegeneration makes it a target for therapeutic intervention for cognitive improvement and neuroprotection. However, a number of studies (Kimura et al., Hum. Mol. Genet., 2007, 16(1), 15-23, and Wegiel et al., FEBS J., 2011, 278(2), 236-45 for review) indicate that overexpression of DYRK1A could be a primary risk factor contributing to the enhancement of both amyloidosis and neurofibrillary degeneration seen in DS, but also AD and other neurodegenerative diseases. Moreover, elevated levels of Aβ peptide may upregulate DYRK1A expression and enhance the contribution of overexpressed DYRK1A to neurofibrillary degeneration and beta-amyloidosis.
DYRK1A phosphorylates key players in AD, namely, APP, Tau, presenilin, and septin-4, DYRK1A acts as a priming kinase, allowing its substrates to be further phosphorylated by GSK3, a key kinase in AD.
Elevated Aβ levels detected in the hippocampus of DYRK1A transgenic mice and in the brain of DS and AD patients suggest that DYRK1A overexpression promotes APP cleavage and Aβ production. Recent studies by Ryoo et al. (J. Neurochem., 2008, 104(5), 1333-44) revealed that DYRK1A phosphorylates APP at Thr668 in vitro and in mammalian cells. Elevated levels of phospho-APP are observed in AD, particularly in the hippocampus. The phosphorylation of APP at Thr668 may facilitate the cleavage of APP by BACE1 and gamma-secretase and enhance the production of Aβ (Vingtdeux et al., Neurobiol. Dis., 2005, 20(2), 625-37). Dyrk1A may also contribute to Aβ production by controlling PS1 phosphorylation at Thr(354). Elevated Aβ levels are detected in the hippocampus of DYRK1A transgenic mice and in the brain of DS patients, suggesting that DYRK1A overexpression promotes APP cleavage and Aβ production. Inhibition of DYRK1A may thus be particularly useful for the regulation or reduction of the formation of Aβ peptide and consequently, the reduction of beta amyloid plaque formation on the brain. Accordingly, DYRK1A can be useful for the treatment of AD and other amyloid-related disorders.
The hypothesis that the elevated activity of DYRK1A contributes to the cognitive deficits in Down syndrome and the development of Alzheimer's disease has stimulated interest in DYRK1A as a potential target for therapeutic inhibitors.
The human DYRK1B gene was mapped to chromosome 19 (19q12-13.11) by radiation hybrid analysis (Leder, S., et al., Biochem. Biophys. Res. Commun., 1999, 254(2), 474-9). The amino acid sequences of DYRK1A and DYRK1B are 84% identical in the N-terminus and the catalytic domain but show no extended sequence similarity in the C-terminal region. DYRK1B contains all motifs characteristic for the DYRK family of protein kinases. In addition, the sequence comprises a bipartite nuclear localization motif. DYRK1B is a muscle- and testis-specific isoform of DYRK1A and is involved in the regulation of nuclear functions.
The protein kinase DYRK1B (also referred to as MIRK) mediates survival and differentiation in many tissues. It is believed to be implicated in certain cancers, particularly solid tumors, see Gao J. et al., Cancer Biology & Therapy, 2009, 8:17, 1671-9 (lung cancer cells), Lee, K. et al., Cancer Research, 2000, 60, 3631-7 (colon cancer cells) and Deng, X. et al., Cancer Research, 2006, 66, 4149-58 (pancreatic cancer cells).
A major problem in the treatment of cancer arises from quiescent cancer cells that are relatively insensitive to most chemotherapeutic drugs and radiation. Such residual cancer cells can cause tumor regrowth or recurrence when they reenter the cell cycle. Earlier studies showed that levels of the serine/theronine kinase MIRK/DYRK1B are elevated up to 10-fold in quiescent G(0) tumor cells (Ewton, D. Z. et al., Mol. Cancer Ther., 2011, 10(11), 2104-14 and Friedman E., Sarcoma, 2011, 260757. Epub 2011 Apr. 13). MIRK/DYRK1B uses several mechanisms to block cell cycling, and MIRK/DYRK1B increases expression of antioxidant genes that decrease reactive oxygen species (ROS) levels and increase quiescent cell viability. MIRK/DYRK1B kinase inhibition elevated ROS levels and DNA damage detected by increased phosphorylation of the histone protein H2AX and by S-phase checkpoints. MIRK/DYRK1B kinase inhibitors increased cleavage of the apoptotic proteins PARP and caspase 3, and increased tumor cell kill several-fold by gemcitabine and cisplatin. A phenocopy of these effects occurred following MIRK/DYRK1B depletion, showing drug specificity. MIRK/DYRK1B knockout or depletion had no detectable effect on normal tissue, suggesting that the MIRK/DYRK1B kinase inhibitor could have a selective effect on cancer cells expressing elevated levels of MIRK/DYRK1B kinase (e.g. lung cancer cells, colon cancer cells, pancreatic cancer cells, ovarian cancer cells, osteosarcoma cells, and rhabdomyosarcoma cells).
Two plant compounds, epigallocatechin-gallate (EGCG) and harmine, have been identified as DYRK1A inhibitors in selectivity profiling studies (Bain et al., Biochem., 2003, 371, 199) (Table 1). EGCG was used in cell culture studies to confirm the presumed role of DYRK1A in signalling events and to rescue brain defects of DYRK1A-overexpressing mice (Guedj et al., PLoS ONE, 2009, 4, 4606). A clinical trial was set-up to investigate the clinical benefits and safety of EGCG administration in young adults with DS, to establish short-term EGCG effects (three months) on neurocognitive performance, and to determine the persistency or reversibility of EGCG related effects after three months of discontinued use (http://clinicaltrials.gov/ct2/show/NCT01394796?term=EGCG&rank=3). The first results look promising (M. Dierssen, Journées internationales Jérôme-Lejeune, 24 Mar. 2011, Institut Pasteur, Paris).
Harmine is a β-carboline alkaloid that has long been known as a potent inhibitor of monoamine oxidase A. Harmine displays excellent specificity for DYRK1A as a potent ATP competitive inhibitor among 69 protein kinases (GB2447791A and Brain et al., Biochem., 2007, 408, 297). However, harmine also inhibits DYRK1B (5-fold less efficiently) and DYRK2 and DYRK3 (50-fold less efficiently) and its inhibitory effect on monoamine oxidase clearly limits its use as a DYRK1A inhibitor (Göckler N, et al., FEBS J. 2009, 276(21), 6324-37).
A number of inhibitors of DYRK1A/1B have been developed. Table 1 below provides the structures of such inhibitors and relevant references disclosing the same.
TABLE 1Inhibitors of DYRK1A/1B (when non specified IC50s or Kds are given for DYRK1A)              
However, there remains a need for new potent and selective inhibitors of DYRK1A and/or DYRK1B. DYRK1A inhibitors will be useful to treat subjects with a central nervous system disease or disorder that is mediated by DYRK1A. DYRK1B inhibitors will be useful to treat subjects with cancers since DYRK1B is overexpressed and mediates survival and differentiation in many cancerous tissues.